REVIEW: Papillomaviruses--to Vaccination and Beyond

H. zur Hausen

Received December 19, 2007
High risk human papillomavirus (HPV) types 16 and 18 DNAs were initially
identified in 1983-1984. Subsequently the DNA of several other high
risk HPV types has been identified. HPV 16 is present in more than 50%
of cervical cancer biopsies, and HPV 18 is close to 20%. Some
geographic variations exist in the prevalence of HPV high risk types:
e.g. HPV 45 is more frequently observed in equatorial Africa, whereas
types 58 and 52 have been more often found in East Asia. Molecular as
well as epidemiological studies demonstrate that high risk HPV are
indeed the causative agents for cervical cancer, they are also involved
in other anogenital cancers, and in 25-30% of oropharyngeal carcinomas.
Some of the mechanistic aspects are discussed in this review.
KEY WORDS: papillomaviruses, cervical cancer, vaccination

This manuscript is dedicated to Professor Gary I. Abelev for
his pioneering studies opening the field of cancer biomarkers

The identification of high risk human papillomavirus (HPV) types as
causative agents of anogenital and oropharyngeal cancers paved the way
for the development of preventive vaccines against these widespread
sexually transmitted infections. Two vaccines are presently available,
both directed against HPV 16 and 18 infections, one of them in addition
against the genital wart virus types 6 and 11. Although these vaccines
provide very effective protection in previously non-exposed women, they
do not seem to possess a significant therapeutic effect in already
infected persons. In addition, their price is presently prohibitive for
their application in most resource-constrained countries. Thus, more
affordable vaccines, preferentially not requiring cold chains and
non-invasive application, are desirable in the future. In addition,
group-specific immunogenicity, as observed for some antigenic epitopes
of the L2 structural protein, will hopefully be further explored in
third generation vaccines. The availability of immunogens protecting
against HPV 16 and 18 infections marks the establishment of a first
vaccine directly developed to protect against a major human cancer.

INFECTIONS AND CANCER--GLOBAL ASPECTS

We can presently estimate that between 20-21% of the global cancer
incidence can be linked to infections [1]. This
includes not only viral infections (e.g. Epstein-Barr virus, human
herpes virus type 8, high risk HPV types, hepatitis B and C viruses,
human T-lymphotropic retrovirus), but also bacterial (Helicobacter
pylori) and parasitic infections (Schistosoma haematobium,
Opisthorchis viverenni, Clonorchis sinensis). Among these
infection-linked human cancers, in males H. pylori emerges as
the major contributor, accounting for about 47%, mainly due to the
global high frequency of gastric cancer. In females, the situation
differs substantially, where H. pylori accounts only for 26% of
the infection-linked carcinomas. Here HPV infections play by far the
most dominant role, contributing to more than 51% of these cancer
cases, whereas HPV in males is responsible for slightly more than 4% of
infection-linked cancer cases.

There exist also major geographic differences in the incidence of
infection-caused human cancers [2]: whereas in
Sub-Saharan Africa and in some East Asian regions up to 40% of all
cancers are linked to infectious events, in Europe and Northern America
less than 10% can presently be linked to infections.

Immunosuppression induced by infections with human immunodeficiency
viruses HIV 1 and 2 may result in activation of persisting tumor
viruses, like Epstein-Barr virus or human herpes virus type 8. The high
prevalence of HIV infections in Sub-Saharan Africa emerges as the
trigger for the presently excessively high rate of Kaposi's sarcomas in
this region, caused by activated human herpes virus type 8 [3].

PAPILLOMAVIRUSES AND CANCER: HISTORICAL DEVELOPMENTS

The infectious nature of human warts was initially described in 1907 by
Ciuffo in Italy [4]. A first link between
papillomatous lesions and cancer was reported for a rare hereditary
disease, epidermodysplasia verruciformis, in 1922 [5]; the contribution of papillomavirus infections in
the causation of these lesions was proven much later [6-8].

Since 1968, cervical cancer was suspected to be linked to human
Herpes simplex virus (HSV) type 2 infections (reviewed in [1]). Our own negative attempts to find HSV-2 DNA in
cervical cancer biopsies prompted considerations to search for other
candidate agents involved in this cancer, whose epidemiology suggested
the involvement of a sexually transmitted factor. Thus, we started in
1972 experimental studies to analyze the possible involvement of human
papillomavirus in this malignancy. In view of some reports claiming the
rare conversion of genital warts into malignant tumors, the virus
supposedly present in genital warts appeared to us as the prime
candidate. This was hypothesized in several publications [9-11]. In the 1970s, two groups
almost simultaneously established the plurality of the HPV family [12, 13] containing by now far
more than 100 well characterized members [14].
Between 1980 and 1982, we published the isolation and characterization
of two HPV DNAs from genital warts and laryngeal papillomas [15-17]. Although we did not find
this DNA in cervical malignancies, using these probes under conditions
of low stringency hybridization permitted initial cloning and
characterization of HPV 16 and 18 DNA directly from cervical cancer
biopsies [18, 19] and from
precursor lesions of anogenital cancer [20].

Subsequent studies performed in a number of laboratories demonstrated
the DNA of these two types in approximately 70% of all biopsies tested.
In addition, a large number of further types found later in some
biopsies eventually resulted in the detection of HPV DNA in virtually
all cervical cancer biopsies carefully analyzed. Mainly HPV 16, but
also HPV 18 and 31, or 33 DNA were also found in other anogenital
cancers and in 25-30% of oropharyngeal carcinomas (reviewed in [1]).

MECHANISTIC ASPECTS OF MALIGNANT CONVERSION OF CELLS INFECTED BY
HIGH RISK HPV TYPES

Several infectious agents act as indirect carcinogens either by inducing
immunosuppression resulting in the activation of other persisting
tumorviruses or by prevention of apoptosis, permitting damaged cells
the continuation of proliferation [1]. High risk
papillomaviruses represent typical direct carcinogenic factors. Two
viral early genes E6 and E7 are not only required for the
development of precursor lesions of cervical cancer, but also necessary
for maintaining the malignant phenotype of cervical cancer cells
(reviewed in [1]). Their silencing in cervical
carcinoma cells commonly results in apoptosis or senescence. In the
stepwise progression from early infection to invasive cancer, regularly
spanning a period of 15-25 years, the individual steps are
characterized by an increase in E6/E7 gene activity within
infected proliferating cells. E6/E7 expression is most
pronounced in carcinomata-in-situ and in invasive cancer.

Progression is accompanied by modifications in genes involved in host
cell signaling cascades. The inability to present viral antigenic
epitopes at the cell surface is mediated by alterations within the HLA
class I pathway and seems to account for the persistence of HPV
infections over periods for more than 2 years in close to 10% of
infected women, whereas the remaining infections are cleared by
immunological interferences within this time period (reviewed in [21]). Two additional mutational events apparently
contribute to further steps in the progression: one involves
modifications in the regulation of the cyclin-dependent kinase
inhibitors p16INK4 [22] and
p14ARF [23]. These proteins exert a
functional control of viral oncoproteins in normal proliferating cells.
The inhibitory effect of p16INK4 for cell proliferation is
blocked by E6. This is circumvented by E7 functions, which stimulate
directly cyclins E and A. p14ARF negatively interferes with
E7 via p53 activation [24]. This is blocked by E6
expression and the resulting degradation of p53. Thus, both viral
oncogenes interact synergistically with each other. The loss of this
second regulatory cascade by the modification of specific cellular
genes seems to correspond clinically to the development of low grade
squamous intraepithelial lesions (LSIL) and an immortalized
growth of such cells under tissue culture conditions.

A third transcriptional control is exerted by a paracrine mechanism and
obviously results from the excretion of specific cytokines, mainly
tumor necrosis factor alpha, from activated macrophages [25, 26]. In HPV infected cells,
which are still able to proliferate, this interference mechanism
suppresses the majority of viral transcripts. In later stages of
progression, the loss of this control is accompanied by a high
expression level of viral oncoproteins. Clinically this corresponds to
the development of high grade squamous intraepithelial lesions (HSIL),
carcinoma in situ, and subsequently, probably by additional
genomic modifications, to invasive carcinoma with metastasis formation.
A scheme of this mode of progression is shown in Fig. 1. The individual steps are schematically outlined in
Fig. 2.

Fig. 1. The development of cervical cancer after primary
infection commonly takes between 15 and 25 years. The events are
schematically outlined in this figure. After primary infection,
individual clones develop escaping from existing extra- and
intracellular control mechanisms acting against uninhibited expression
of viral oncogenes within proliferating cells. In the course of these
developments E6/E7 oncogene expression increases substantially.
The viral oncoproteins contribute effectively to chromosomal
instability and aneuploidy.

Fig. 2. Schematic representation of control mechanisms blocking
viral oncoproteins of viral RNA transcription (a). Viral DNA
persistence seems to occur after mutational events within the HLA class
I pathway (b). The interference with the pathway blocking the function
of viral oncoproteins results in early lesions (c) and seems to
correspond in tissue culture to the state of immortalization. The
eventual disappearance of a paracrine transcriptional control is
correlated by a high rate of E6/E7 oncoprotein expression and the
development of high grade lesions (carcinoma in situ) (d).
Further molecular events are probably required to result in invasive
growth of the lesions.

Two key observations in understanding the function of E6 and E7
oncoproteins were made in 1989 [27] and 1990 [28, 29]. The discoveries by
Dyson et al. in 1989 of an interaction of E7 with pRb and by Werness et
al. and Scheffner et al. in 1990 of E6 interacting and degrading p53
permitted molecular approaches to study functions of viral oncoproteins
and to contribute to basic understanding of viral carcinogenesis. In
both instances, these interactions result in the degradation of the
respective cellular proteins (reviewed in [1]). In
particular, the formation of complex between E6 and the E6/E3 ubiquitin
ligase has consequences for a number of intracellular pathways: it
results in the activation of telomerase, the degradation of several PDZ
proteins regulating intracellular pathways, and the activation of
cyclin D/cdk4/6 complexes. The latter is counteracted by the high
induction of p16INK4 as a consequence of E7/pRb interaction
followed by the degradation of pRb. A number of further interactions of
viral oncoproteins with host cell components have been described in
recent years, which will not be reviewed here.

VACCINATION AGAINST HPV TYPES

Accumulating data on the immunological control of persisting high risk
HPV infections raised early hopes for the prevention of cervical cancer
by vaccination [30]. The interest of
pharmaceutical companies in the production of an HPV vaccine arose only
when epidemiological studies were published supporting the existing
experimental evidence [31].

Presently available vaccines are based on virus-like particles [32, 33], initially produced by
inserting the L1 and L2 open reading frames of HPV 16 into vaccinia
virus vector systems, subsequently by exclusively expressing L1 in
yeast cells. The expression of the L1 open reading frame, coding for
the major capsid protein, results in the formation of empty capsid
structures, virus-like particles (VLP). After purification and addition
of specific adjuvants, these VLPs are being used for vaccination. Two
vaccines are presently available and in clinical use: one contains VLPs
of HPV 6, 11, 16, and 18, the other only HPV 16 and 18. Since HPV 16
covers slightly more than 50% of all cervical cancers and HPV 18 close
to 20%, it is anticipated that these vaccines should protect against at
least 70% of cervical carcinomas and their precursor lesions. Recent
data also indicate that there exists a certain cross-protection against
the HPV 16-related type HPV 31, and the HPV 18-related type HPV 45,
which may bring up the rate of protection to close to 80%.

The available data covering by now more than five years of follow-up are
remarkably impressive. Both vaccines appear to be highly efficient in
preventing infections by the respective HPV types in previously
non-exposed women [34, 35].
The vaccines do not exhibit a significant protective effect in already
infected women, thus they are recommended to be applied to girls and
young women in age groups between 9 and 25 years.

The vaccines are proposed to be intramuscularly injected in three shots
at months 0, 1 or 2, and 6. The antibody conversion occurred in
approximately 100% of vaccinated persons. Antibodies usually persisted
at high titers for up to 6 years, whereas the titers were commonly low
or negative in placebo controls.

Approximately 83% of all cervical cancers occur in resource-constrained
countries in Sub-Saharan Africa, Central and South America, and South
East Asia. The present high costs of the vaccines, ranging from US $360
in the United States to about 500 Euro in European countries are
prohibitive for a global application specifically in those countries
that most badly need it.

A number of additional questions still require further exploration. This
concerns in particular the age groups, which should be vaccinated. The
recommendations vary between individual countries, usually in the range
of 9-25 years. It is likely, however, that even persons infected with
one or two of the virus types present in the vaccine will profit from
the vaccination by acquiring protection against additional types
present in the vaccine. In view of the ambiguities of negative PCR or
antibody results, it is difficult to come up with a uniform proposal.
Clearly, in counties without sufficient screening and gynecological
control of women, vaccination should start early prior to the onset of
sexual activities.

Two other aspects deserve consideration: should the vaccine also be
applied to boys and young adult males, and does vaccination result in a
prolongation of intervals for cervical cancer screening?

Vaccination of boys and young adult males is often not considered to be
desirable. There exist, however, several reasons to consider this group
also in vaccination strategies. Two types of high risk HPV-linked
cancers occur even more frequently in males than in females. This
accounts for anal and perianal cancers and for 25-30% of oropharyngeal
cancers. In addition, genital warts occur at relatively high frequency
in both sexes. At least one of the vaccines protects against
approximately 90% of these infections. A further aspect is the
consideration of gender solidarity, since high risk HPV are transmitted
from males to females and vice versa. Although controlled clinical
trials after vaccination of boys have not yet been published, it is
highly likely that their reactivity will not significantly differ from
that of girls.

Successful application of the presently available vaccines should not
result in diminished screening of women. The present vaccines do not
protect against other high risk types which may still account for
20-30% of cervical cancers. Thus, a follow-up of early cervical lesions
and their removal in case of progression will remain mandatory.

Besides these general aspects, some global perspectives of HPV
vaccination should be considered. The remaining major problem is the
question how can we achieve a global application of the vaccine? This
is of course very closely linked to a drastic reduction in the price
for the vaccine. But it also involves the mobilization of the political
will, necessary regulatory actions, effective delivery, and sufficient
financial support. In addition, careful studies are still missing,
indicating a possible protective effect of only two injections. There
exists the problem of creating a global awareness for the importance of
cervical cancer and for sustained information of local physicians,
health workers, politicians, school teachers, and company employees.
How can we develop suitable management structures for a global
application of this vaccine? The involvement of the World Health
Organization, the United Nations, and of a large number of NGOs seems
to be mandatory. Thus, we are only in the beginning of hopefully
solving an important global cancer problem. Cervical cancer still ranks
number two in the global cancer incidence of women with close to
500,000 women acquiring this disease annually and approximately one
half of them succumbing to this cancer.

FUTURE PERSPECTIVES

Several additional alternatives exist for future generations of
vaccines. The foremost question will be the provision of affordable
vaccines at a price permitting their application in regions of the
developing world. This will require new concepts in vaccine production.
Some of them are already presently being explored. This concerns mainly
the production of vaccines in bacteria. This commonly does not seem to
lead to VLP production, but only in capsomer assembly. Yet, these
capsomers are immunogenic and could replace VLP vaccines, albeit
probably at higher protein concentrations.

A number of alternative concepts are also under consideration. The
N-terminal part of the L2 protein induces a broad range of
group-specific immunity, resulting in neutralizing antibodies for a
large number of HPV types. This is the basis for anticipating the
future availability of a vaccine covering a large spectrum of
papillomavirus infections. The present problem for an application
exists in the relatively low immunogenicity of the respective L2
antigenic epitopes. Other concepts involve the application of modified
naked viral DNA, viral RNA vector systems that may turn out
to be useful for prophylactic and therapeutic vaccinations, or
intranasal application of genetically modified attenuated Salmonella
strains expressing HPV proteins. These opportunities have not yet
been tested extensively.

One of the more promising concepts is the application of
adeno-associated virus (AAV) vector systems carrying the gene for HPV
L1 proteins. This system has the advantage of being relatively
heat-resistant (AAV tolerate temperatures of up to 60°C), thus it
does not require a cold chain. In addition, these preparations may be
applied as intranasal sprays, avoiding injections. A first preclinical
test revealed the immunogenicity and efficacy of this vaccination after
intranasal application in mice [36].

In spite of the very remarkable progress made in the prevention of
specific high risk HPV infections, several problems remain to be
resolved: they concern the inclusion of other high risk types into the
vaccine preparations and hopefully the eradication of all types engaged
in cancer of the cervix. Of course, the most important problem to be
resolved remains the production of an affordable vaccine for developing
parts of the world.

Other problems requiring urgent attention are the immunotherapeutic or
chemotherapeutic interference with persisting infections, early and
late cervical intraepithelial neoplasias, carcinomas in situ,
and invasive cancer. At this moment, it seems that immunotherapy may
have a chance in interfering with viral persistence and very early
lesions. This remains, however, to be seen in future studies. Targeted
chemotherapy is a promising area of research. The three dimensional
structure of various viral oncoproteins is now available. This may
provide hope for successful future interventions.

There still exist other modes of intervention by small interfering RNAs,
where at least in tissue culture experiments shut-off of E7 functions
in cervical cancer cells results in apoptosis [37]. In addition, other non-coding RNAs, as
exemplified by viral and cellular microRNAs, may fulfill important
functions in the positive or negative regulation of viral oncogenes.
Some of those may also turn out to be useful for therapeutic
interferences. A major question still to be resolved remains the mode
of application of RNA-based pharmaceuticals. Hopefully, new concepts
will arise along those lines in forthcoming years.